The invention relates to DNA vectors.
Mammalian cell expression vectors based on DNA viruses have been widely discussed as gene delivery vehicles for genetic therapy. Among the different DNA viruses proposed for this purpose have been adenoviruses, baculovirus, Epstein Barr virus, and herpes simplex virus. In addition other smaller viruses that have an intranuclear phase in which the viral genome is present as a double stranded DNA, such as retroviruses and parvoviruses, have been proposed as gene delivery vehicles.
Adenoviral vectors (AdV), for example, have a recognized potential for gene delivery, founded in their broad host range, robust growth in culture, and capacity to infect mitotically quiescent cells (Graham and Prevec, Manipulation of adenovirus vectors, p. 109–128, In E. J. Murray (ed.), Methods in Molecular Biology, vol. 7, Humana, Clifton, N.J., 1991; Trapnell and Gorziglia, Curr. Opin. Biotechnol. 5:617–625, 1994). AdV can be propagated in a helper cell line, 293, a human embryonic kidney cell line transformed by adenovirus type 5 (Graham et al., J. Gen. Virol. 36:59–72, 1994). 293 cells express the viral E1 gene products (E1a and E1b) that are the master regulatory proteins for subsequent viral gene expression. E1 deleted viruses can propagate in 293 cells, but not in other cells. Although it would be expected that E1 deleted viruses lack the machinery to express viral genes, several studies have demonstrated that cellular E1-like components can stimulate viral gene expression (Imperiale et al., Mol. Cell. Biol. 4:867–74, 1984; Onclercq et al., J. Virol. 62:4533–7,1988; Spergel et al., J. Virol. 66:1021–30, 1992). The expression of these viral genes results in the relatively rapid elimination of transduced cells in vivo as a result of cytotoxic T cell responses (Yang et al., Immunity 1:433–42, 1994; Yang et al., Gene Ther. 3:137–44, 1996; Yang et al., J. Virol. 69:2004–15, 1995).
Thus attention has been focused on eliminating the remaining vestiges of viral expression. Viral genes that have been deleted for this purpose include the gene for E4 proteins (Armentano et al., Hum. Gene Ther. 6:1343–53, 1995; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731–6, 1996; and Yeh et al., J. Virol. 70:559–565, 1996), DNA binding protein (Engelhardt et al., Proc. Natl. Acad. Sci. USA 21:6196–6200, 1994; and Gorziglia et al., J. Virol. 70:4173–8, 1996), DNA polymerase (Amalfitano et al., J. Virol. 72:926–33, 1998), and the preterminal protein (Schaack et al., Proc. Natl. Acad. Sci. USA 93:14686–91, 1996). The most aggressive approach has been the creation of helper virus-dependent vectors that lack all viral genes (Hardy et al., J. Virol. 71:1842–9, 1997; Kochaneketal., Proc. Natl. Acad. Sci. USA 93:5731–6, 1996; Lieber et al., J. Virol. 70:8944–60, 1996; Mitani et al., Proc. Natl. Acad. Sci. USA 92:3854–8, 1995; and Parks et al., Proc. Natl. Acad. Sci. USA 93:13565–13570, 1996). These vectors have high capacity, evoke reduced cellular immune responses and show prolonged expression in vivo (Morsy et al., Proc. Natl. Acad. Sci. USA 95:7866–71, 1998). However to deploy these viruses on the scale required for human clinical application presents major challenges because a cesium chloride (CsCl) gradient is needed to remove the helper virus.